The present invention relates to a diagnostic device with integrated photodetector, and to a diagnostic system including said diagnostic device.
As is known, available today are numerous diagnostic devices for biological applications.
In general, diagnostic devices each include a respective assay. In turn, the assay may comprise a solid substrate, which is typically of a flat type and has a surface functionalized so as to present detection regions, inside which receptors, which are provided with specific markers, are immobilized.
In practice, by “receptor” is meant any member of a pair or of an N-tuple of elements that can bind together. Consequently, each receptor is able to mate, or in any case react, with a respective binding mate, or else with a respective plurality of binding mates, enabling detection thereof. For example, receptors can comprise biomolecules (DNA, RNA, proteins, antigens, antibodies, haptens, sugars, etc.) or chemical species, or micro-organisms or parts thereof (bacteria, viruses, spores, cells, etc.). Binding mates are also known as “targets” or “target molecules”; moreover, receptors are also known as “probes” or “probe molecules”.
As regards markers, each one of them is such that, when the corresponding receptor mates or interacts with its own binding mate, or binding mates, it can be activated, i.e., it can mate with the receptor by mating with the binding mate. Given any receptor, it is hence found that, when it is in direct contact with a specimen to be analysed, present inside which are binding mates that are able to mate/interact with the receptor, the corresponding markers can be activated.
In particular, in so-called fluorescence diagnostic devices, an activated marker, if excited with a light radiation at a certain wavelength λe, emits a light radiation of its own having a wavelength λf longer than the wavelength λe. In general, said markers are known as “fluorescence markers”.
This being said, there are known, among other things, the so-called “three-component binding assays”, which each use a first immobilization of a first antibody on a solid substrate, said first antibody being able to mate with an antigen present in a standard solution. Binding with the antigen is then detected thanks to a second antibody, which functions as marker and binds to an epitope of the antigen itself. The second antibody has a fluorescent label attached to it; consequently, the amount of fluorescence is correlated to the amount of antigens present in the standard solution.
In practice, by detecting the light radiation at the wavelength λf, information regarding the chemico-physical characteristics of the specimen to be analysed may be inferred since the light intensity detected is a function of the amount of activated markers in the assay, and hence of the amount of molecules or biomolecules detected by the assay. For this purpose, the optical detector is sensitive to the wavelength λf of the light radiation emitted by the markers.
In greater detail, in order to detect the light radiation emitted by the markers, there is known the use of optical detectors, which enable scanning of small portions of specimens to be analysed in order to determine characteristics and/or properties of the specimens. These optical detectors usually comprise optical elements and movement devices having particularly small dimensions, for example in the region of a few millimeters. In addition, optical detectors are known in which the movement devices are formed by micro-electromechanical systems (MEMS).
Irrespective of the details of implementation, typically a generic optical detector is coupled to a corresponding diagnostic device, set inside which is at least one specimen to be analysed in such a way that, by illuminating with a first beam of light at least one portion of the specimen, the optical receptor receives a second beam of light coming from the specimen itself and generated, in the case of a fluorescence diagnostic device, by excitation of the specimen by means of incidence of the first beam of light.
In order to increase the capacity of analysis and, in particular, to be able to detect ever more limited amounts of activated markers, it is desirable to maximize the light intensity of the beam emitted by fluorescence and/or increase the sensitivity of the optical detector.
By way of example, the patent application No. TO2010A000437 filed on May 25, 2010 in the name of the present applicant describes an optically accessible microfluidic diagnostic device, which can hence be coupled to an optical detector.
The diagnostic device comprises a substrate of semiconductor material, overlaid by a compatible layer, made, for example, of silicon dioxide (SiO2).
The diagnostic device described in the patent application No. TO2010A000437 further comprises a structural layer, formed, for example, by a photoresist and arranged on top of the compatible layer, with which it is in direct contact. Formed within the structural layer are one or more channels, obtained by means of selective removal of one or more portions of the structural layer, until the underlying compatible layer is reached and exposed. The bottom of each channel is hence delimited by the compatible layer, whilst the side walls are delimited by the structural layer. Each channel houses one or more detection regions, where receptor biomolecules are present, deposited for example by means of the so-called “automated spotting technique”. The detection regions are hence formed by drops of biological material, said drops having dimensions comprised between a few picoliters and a few microliters. Access to each channel and to the corresponding detection regions is provided by a corresponding inlet hole and by a corresponding outlet hole, which extend through the substrate and the compatible layer. With regard to the compatible layer, it is in fact defined “compatible” because it does not interfere with the detection regions, and in particular with the receptors present therein. Optionally, the compatible layer can be replaced by a non-compatible layer, provided that it is appropriately passivated. Moreover, the compatible layer can be functionalized, for example by means of addition of hydroxyl groups (OH groups) in order to enable immobilization of the receptors.
The diagnostic device described in the patent application No. TO2010A000437 further comprises a cover layer, formed by an adhesive film and laid on the structural layer, with which it is in direct contact so as to seal the channels at the top. In use, the excitation radiation is generated above the cover layer and impinges upon the detection regions after traversing the cover layer. The fluorescence radiation, generated by the excited markers, is in turn detected by means of a photodetector set on top of the cover layer, hence after prior traversal of the cover layer, which is hence transparent both to the wavelength λe and to the wavelength λf.
The compatible layer is also transparent to the wavelength λe and to the wavelength λf. Furthermore, in order to increase the light intensity of the beam emitted by fluorescence, the thickness of the compatible layer is sized in such a way that the corresponding optical thickness is substantially equal to an integer and odd multiple of the wavelength λe of the excitation radiation, the direction of incidence of which on the compatible layer is assumed as being orthogonal to said layer. In this way, thanks to phenomena of constructive interference, there is an increase of the electrical field associated to the excitation radiation in the detection regions and, consequently, a maximization of the light radiation emitted by the excited markers.
Even though the geometry adopted in the patent application No. TO2010A000437 envisages that the optical source, which generates the excitation radiation, and the optical detector are set on one and the same side with respect to the channel, and hence with respect to the assay, there are also known diagnostic systems that adopt different geometries.
For example, diagnostic devices are known of the type described in the patent application No. WO2007/144864, where a detection region is present, which is carried by a support and is set between the optical source and the optical detector.
Furthermore, according to the patent application No. WO2007/144864, the optical detector is a Geiger-mode avalanche photodiode (GM-APD), also known as single-photon avalanche diode (SPAD) in so far as it is able to detect single photons.
In general, a SPAD is formed by an avalanche photodiode and hence comprises a junction, typically of P+/N type, or else an N+/P junction.
In greater detail, the junction has a breakdown voltage VB and is biased, in use, with a reverse-biasing voltage VA higher in modulus than the breakdown voltage VB of the junction, typically 10-20% higher. In this way, generation of a single electron-hole pair, following upon absorption of a photon impinging upon the SPAD, is sufficient to trigger an ionization process that causes an avalanche multiplication of the carriers, with gains of the order of 106 and consequent generation in short times (hundreds of picoseconds) of the avalanche current. This avalanche current can be conveniently collected, typically by means of an external circuitry connected to the junction, for example by means of anode and cathode contacts, and forms an electrical signal at output from the SPAD.
The gain and the likelihood of detecting a photon, i.e., the sensitivity of the SPAD, are directly proportional to the value of reverse-biasing voltage VA applied to the SPAD. However, the fact that the reverse-biasing voltage VA is appreciably higher than the breakdown voltage VB means that the process of avalanche ionization, once triggered, is self-sustaining. Consequently, once triggered, the SPAD is no longer able to detect photons, with the consequence that, in the absence of appropriate remedies, the SPADs described manage to detect the arrival of a first photon, but not the arrival of subsequent photons. To be able to detect also these photons, it is necessary to quench the avalanche current generated within the SPAD, arresting the process of avalanche ionization. In practice, it is necessary to reduce, for a period of time known as “hold-off time”, the effective voltage Ve across the junction, this effective voltage Ve coinciding with the reverse-biasing voltage VA only in the absence of photons, i.e., in the absence of current within the SPAD. In this way, the ionization process is inhibited, and the avalanche current is quenched. Next, the initial conditions of biasing of the junction are restored in such a way that the SPAD is again able to detect photons.
In order to reduce the effective voltage Ve across the junction subsequent to absorption of a photon, SPADs adopt the so-called “quenching circuits”, either of an active type or of a passive type.
Irrespective of the details of implementation of the SPAD, and thanks to the use of the latter, the diagnostic system described in the patent application No. WO2007/144864 is characterized by a high sensitivity; however, it calls for particular care in use. The diagnostic system is operated as shown in FIGS. 1a and 1b. 
In particular, as shown in FIG. 1a, the optical source operates in pulsed regime; i.e., it generates pulses of the excitation radiation, each pulse having a duration for example in the region of a few microseconds. Purely by way of example, FIG. 1a plots a supply voltage of the optical source; hence, this is also indicative of the intensity of the excitation radiation emitted by the optical source.
Considering a single pulse of the excitation radiation, and in the case of presence of activated markers in the detection region, they are excited by this pulse and emit light radiation at the wavelength λf. If the radiation emitted by the activated markers and excited is referred to as “optical response signal”, this decays exponentially in time, with a time constant comprised between a microsecond and a few milliseconds.
In detail, the geometry of the diagnostic system described in the patent application No. WO2007/144864 is such that, if the optical signal to be detected is referred to as “optical signal that impinges upon the optical detector”, this is formed both by the excitation radiation and by the optical response signal emitted by the activated markers.
After the optical detector has been impinged upon by the optical signal to be detected, it generates an electrical signal of the type shown in FIG. 1b. In particular, the electrical signal has a first portion and a second portion, which follow one another in time and correspond, respectively, to a first portion and a second portion of the optical signal to be detected.
The first portion of the optical signal to be detected is due principally to the pulse generated by the optical source, whilst the second portion is caused by the light radiation emitted by the activated markers and excited and consequently has a decreasing-exponential evolution.
This being said, during the first portion of the optical signal to be detected, the latter has an intensity such as to cause a sort of blinding of the optical detector; i.e., it causes saturation thereof. In other words, the first portion of the electrical signal is characterized by a high intensity, which is substantially independent of the number of activated markers and hence does not contain information useful for diagnostic purposes. Consequently, the optical detector is controlled in such a way that only the second portion of the electrical signal generated thereby is effectively processed for diagnostic purposes.
This modality of control of the optical source and of the optical detector hence exploits the so-called “phenomenon of delayed fluorescence” and enables benefiting from the advantages associated to the use of the SPAD, and moreover does not require a narrow-band optical filter having the function of filtering the excitation radiation to be set between the specimen to be analysed and the optical detector. However, in order to excite as many markers as possible, the intensity of the pulses of the excitation radiation is particularly high. Consequently, these pulses cause, once received by the SPAD, formation of a large number of electrical carriers within the semiconductor body that forms the SPAD itself. In turn, the electrical carriers cause an increase in the so-called “dark noise”.
In detail, the SPAD operates in a way such as to determine, for each time interval, a corresponding count, i.e., a corresponding number of photons received thereby during said time interval. This being said, the aforementioned electrical carriers cause an increase in the so-called dark counts, namely, counts that are in any case determined by the SPAD in the absence of the optical signal to be detected. In practice, the electrical carriers hence lead to an increase of the count supplied by the SPAD as compared to the photons effectively received during the time interval.
In greater detail, the increase in dark counts occurs not only in coincidence with reception of the pulse, i.e., during the aforementioned first portion of the optical signal to be detected, but continues even after the pulse is exhausted; i.e., it continues even during the aforementioned second portion of the optical signal to be detected.
Purely by way of example, FIGS. 2a-2c show the time plot of the electrical signal at output from the SPAD, on the hypothesis of absence of activated markers. Moreover, FIGS. 2a and 2b refer to the cases of pulses having equal intensity and duration respectively of 100 ns and 2 ns, whilst FIG. 2c regards the case of absence of pulses. In practice, the plot shown in FIG. 2c has a certain number of peaks P per unit time, this number being indicative of the intrinsic “dark noise” of the SPAD. The plots shown in FIGS. 2b and, above all, 2a show, instead, an increase of the number of peaks P per unit time. In each of these two cases, this increase is due to the reception, by the SPAD, of a corresponding pulse PP generated by the optical source.
The increase in the dark noise is proportional to the energy of the pulses and can hence be contained by means of limitation of the latter, but this entails a reduction of the number of activated markers that are effectively excited.
The aim of the present invention is to provide a diagnostic device that will enable the drawbacks of the known art to be overcome at least in part.